Genome Sequence of a Cluster CR2 Gordonia terrae Phage, StarStruck

ABSTRACT Bacteriophage StarStruck is a lytic Siphoviridae phage that infects Gordonia terrae 3612. The 68,128-bp genome of StarStruck has a GC content of 65.4% and contains 92 protein-coding genes, including the gene for a HicA-like toxin. StarStruck was assigned to subcluster CR2 based on >35% shared gene content with other cluster CR genomes in the Actinobacteriophage Database.

A ctinobacteriophages, which are viruses that infect bacteria of the phylum Actinobacteria, are extremely abundant and diverse (1)(2)(3)(4). By characterizing lytic bacteriophages, we increase our understanding of phage diversity and susceptibility, which is relevant to applications such as phage therapy and biological control of foaming bacteria in wastewater treatment plants (5,6). StarStruck was isolated from a soil sample collected at 27°C in Orono, Maine (44.896455N, 68.66614W), using Gordonia terrae 3612 (7). Soil extracts were prepared in peptone-yeast extract-calcium (PYCa) medium and filtered on 0.22-mm filters before inoculation with G. terrae and incubation at 30°C for 2 days. The extract was diluted and plated onto PYCa agar in soft agar containing G. terrae, and plaques were purified by four rounds of plaque assays (7). After incubation for 2 days at 30°C, StarStruck formed 4-mm clear plaques with turbid edges on a lawn of G. terrae, but lysogen isolation techniques did not yield stable lysogens (7). The particle morphology of StarStruck was determined by negative staining transmission electron microscopy. StarStruck has a Siphoviridae morphology with a 65-nm (standard error [SE], 63.2 nm) icosahedral head and a 297.5-nm (SE, 67.5 nm) flexible, noncontractile tail (n = 3 particles).
DNA was extracted from a high-titer lysate by phenol-chloroform extraction and prepared for sequencing using the NEBNext Ultra II library preparation kit (New England BioLabs, Ipswich, MA) (8). Sequencing on an Illumina MiSeq platform yielded 561,846 single-end 150-bp reads. Newbler v2.9 and Consed v29 (9) were used to assemble raw reads and check for completeness, yielding a 68,128-bp genome with a GC content of 65.4%. Genome ends are defined by single-stranded 10-bp 39 extensions (CGCCGCGTAC). StarStruck shares .35% gene content with members of cluster CR in the Phamerator database Actino_Draft and was assigned to subcluster CR2 (4, 10, 11).
Like many cluster CR phages, StarStruck encodes a HicA-like toxin (gp7) within the 12,000-bp region separating the small-and large-subunit terminases. Another interesting feature of StarStruck is the location of the lysin B (gp19) within this region, rather than adjacent to the lysin A genes (protease C39 domain [gp49] and glycosyl hydrolase domain [gp50]), which are located downstream of the minor tail proteins.
Data availability. StarStruck is available at GenBank with the accession number ON456333 and the Sequence Read jhu7 (SRA) accession number SRX14816101.

ACKNOWLEDGMENTS
This research was made possible by the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program of the Howard Hughes Medical Institute. We thank Daniel Russel and Rebecca Garlena for sequencing services and assembly of the StarStruck genome. We are grateful to Geoff Williams at the Brown Bioimaging Facility for providing electron microscopy services. Students are FIG 1 Genome map of Gordonia phage StarStruck. The ruler indicates genome coordinates in units of kilobase pairs. Forward and reverse genes are represented by colored boxes above and below the ruler, respectively. Genes were assigned to a phamily using Phamerator (10) with the Actino_Draft database, and different phamilies are indicated by different colors. An electron micrograph of StarStruck is shown in the inset, with a scale bar of 100 nm. The average diameter and length of the particle head and tail were determined by measuring the dimensions of three different particles. grateful for the support of teaching assistants Remi Geohagen, Mathew Cox, Allie Conner, Caitlin Wiafe-Kwakye, Abigail McNally, and Jacob Cote.
Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant P20GM103423.